Advances in microscopy have permitted increasingly sophisticated investigations of biological and other systems. In traditional microscopy, a specimen is illuminated with a broadband (“white”) light source, and a magnified image of the specimen is produced. Resolution and image contrast in such systems generally depend on specimen spectral absorbance and microscope objective numerical aperture and aberrations. Microscopy can also be based on radiation produced in a specimen in response to a exposure to a suitable light flux. For example, in fluorescence microscopy, fluorescence produced in a specimen in response to a stimulating light flux is used to form a specimen image. In other examples, a specimen can emit a light flux based on a stimulating light flux via a multiphoton process. In both fluorescence microscopy and multiphoton microscopy the emitted light flux can be used to produce an image directly, or the emitted light flux can be localized by scanning the stimulating light flux across the specimen.
While microscopy-based specimen analysis based on fluorescence or multiphoton processes can provide significant specimen information, such analysis is typically hampered by inefficiencies in the fluorescence collection and multiphoton excitation processes. In some investigations, low efficiencies can be compensated for by increasing the time during which the specimen is exposed to the stimulating light flux, resulting in longer measurement times. In other examples, the stimulating light flux produces specimen changes so that increased exposures cannot be used. Therefore, improved microscopy methods and apparatus are needed.